1. Technical Field
The field of this invention is the polymerase chain reaction.
2. Background of the Invention
The polymerase chain reaction (PCR), in which a polymerase (typically a thermostable polymerase) is used to produce amplified amounts of nucleic acids from an initial template nucleic acid, finds use in a number of diverse research, clinical, forensic and industrial applications.
One application where PCR finds use is in the study and modification of wild-type protein function by error-prone PCR mutagenesis (Leung et al., infra; Cadwell and Joyce, infra). In error-prone PCR, a gene of interest is amplified by PCR under buffer conditions which promote the random misincorporation of nucleotides. The pool of mutant genes from the error-prone PCR reaction is then cloned and used as a library to search for altered protein function.
In the past, the study of the types of mutations occurring in error-prone PCR has relied directly on DNA sequencing. The high cost and cumbersome work associated with sequencing large numbers of clones has limited the extent of experimentation with respect to error-prone PCR and mutational studies in general. As alternatives to sequencing, several phenotypic assays have been developed which detect mutations by observing losses in streptomycin resistance (Mo et al., infra), changes in the function of the lac operon (e.g. blue/white screening)(Lundberg et al., infra; Barnes, infra), and by reversion of a green fluorescent protein mutant (Cariello et al., infra). In addition, gel-based assays have been developed which include single-strand conformation polymorphism (SSCP) (Brail et al., supra) and denaturing gradient gel electrophoresis (DGGE) (Cariello and Skopek, infra).
While these assays can provide accurate information about the relative extents of mutation occurring in a homogenous gene population, they provide little information about the types of mutations occurring. In addition, the gel-based assays are inherently dependent on an electrophoretic separation for comparison of mutation rates, and the phenotypic methods all require ligation and cell transformation procedures associated with typical recombinant DNA cloning methods.
As such, there is a need for the development of alternative methods of evaluating the fidelity of a polymerase under a given set of PCR conditions. Of particular interest would be the development of a rapid in vitro assay which can easily quantify and characterize the relative levels of random mutations occurring in error-prone PCR.
Relevant Literature
References of interest include: Barnes, xe2x80x9cThe fidelity of Taq polymerase catalyzing PCR is improved by an N-terminal deletion,xe2x80x9d Gene (1992)112(1):29-35; Brail et al, xe2x80x9cImproved polymerase fidelity in PCR-SSCPA,xe2x80x9d Mutat. Res. (1993) 303(4):171-5; Cadwell et al., xe2x80x9cRandomization of genes by PCR mutagenesis,xe2x80x9d PCR Methods Appl. (1992) 2(1):28-33; Cariello and Skopek, xe2x80x9cMutational analysis using denaturing gradient gel electrophoresis and PCR,xe2x80x9d Mutat.Res. (1993) 288(1):103-112; Cariello et al., xe2x80x9cA novel bacterial reversion and forward mutation assay based on green fluorescent protein,xe2x80x9d Mutat. Res. (1998) 414(1-3):95-105; Day et al. xe2x80x9cNucleotide analogs and new buffers improve a generalized method to enrich for low abundance mutations,xe2x80x9d Nuc. Acids Res. (1999) 27(8): 1819-1827; Fromant et al., xe2x80x9cDirect random mutagenesis of gene-sized DNA fragments using polymerase chain reaction,xe2x80x9d Anal. Biochem. (1995) 224(1):347-53; Halling, xe2x80x9cIs random mutation more rational?xe2x80x9d Nat. Biotechnol. (1996) 14(4):432, 436; Leung, xe2x80x9cA method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction,xe2x80x9d Technique (1989) 1(1): 11-15; Lundberg et al., xe2x80x9cHigh-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus,xe2x80x9d Gene (1991) 108(1):1-6; Mo et al., xe2x80x9cMutational specificity of the dnaE173 mutator associated with a defect in the catalytic subunit of DNA polymerase III of Escherichia coli,xe2x80x9dJ. Mol. Biol. (1991) 222(4):925-36; Sandy et al., xe2x80x9cGenotypic analysis of mutations in Taq I restriction recognition sites by restriction fragment length polymorphism/polymerase chain reaction,xe2x80x9d Proc. Nat""l Acad. Sci. USA (Feb. 1992) 89:890-894; Tyagi and Kramer, xe2x80x9cMolecular beacons: probes that fluoresce upon hybridization,xe2x80x9d Nat. Biotechnol. (1996) 14(3):303-308; and Vartanian et al., xe2x80x9cHypermutagenic PCR involving all four transitions and a sizeable proportion of transversions,xe2x80x9d Nucleic Acids Res. (1996)24(14):2627-31.
Methods of determining the fidelity of a given set of polymerase chain reaction conditions are provided. In the subject methods, a template polydeoxyribonucleotide that includes a pseudo restriction endonuclease site is amplified under the polymerase chain reaction conditions to be evaluated. The pseudo restriction endonuclease site differs from its corresponding true restriction endonuclease site by at least one nucleotide substitution. The resultant amplified product population is then contacted with the corresponding restriction endonuclease and any resultant cleavage products are detected. The detected cleavage products, or absence thereof, are then related to the fidelity of the polymerase chain reaction conditions used to amplify the template polydeoxyribonucleotide. Also provided are kits for practicing the subject methods.